DESCRIPTION: Voltage-dependent Ca2+ channels are crucial for many physiological processes that couple electrical excitation events and cellular functions. Understanding their permeation and gating is crucial for design of drugs for a range of neurological and cardiac disorders, epilepsy, stroke, and hypertension. Work of our laboratory has shown that Ca2+ ions themselves are intimately involved in voltage-dependent gating of Ca2+ channels. Our recent results indicate that during activation the channel's affinity for Ca2+ decreases. When the channel is inactivated, its selectivity properties and binding of inorganic blockers are also changed indicating an increase of the apparent affinity for Ca2+. Although these observations call for a change of the previously held view that gating and selectivity are autonomous aspects of Ca2+ channel functioning, the novel link between gating and permeation does not negate or contradict existing knowledge about Ca2+ channels. It provides additional resolution to the underlying mechanisms and makes specific predictions that are investigated in this proposal. Our general working hypothesis is that modification of the channel's affinity to Ca2+ is a part of the mechanism of gating. We propose to investigate: the link between Ca2+-dependent component of gating charge movement and the increase Ca2+ ion exit rate during activation, the mechanism of Ca2+-specific changes in the accessibility of the selectivity filter during activation (Aim 1);the mechanism of the block of the selectivity filter by Ca2+ ions during Ca2+-dependent inactivation and whether the final effect of the control of inactivation by auxiliary subunits and calmodulin is at the selectivity filter (Aim 2). The proposed studies involve recording of ionic and gating currents at low extracellular Ca2+, analysis of mutants of the selectivity filter, effects of laser-flash photolysis of caged Ca2+ on ionic and gating currents. Results and conclusions from the tests proposed will be incorporated into a quantitative model of activation and inactivation, with the ultimate goal of explaining the integral role of Ca2+ ions in voltage-dependent gating of Ca2+ channels. This mechanistic analysis of causes and effects will be a crucial complement of further structural and functional studies. PUBLIC HEALTH RELEVANCE: This project probes the role of permeating ions in gating of voltage-dependent Ca2+ channels. These channels are the interface between electrical activity in the plasma membrane and intracellular Ca2+ signaling. Understanding their permeation and gating is critical for design of drugs whose actions depend on the state of the channel and therefore are relevant therapeutically for various neurological and cardio-vascular disorders.